Product and process for inhibition of biofilm development

ABSTRACT

Disclosed are compositions and methods for the inhibition of biofilm formation or reduction of existing or developing biofilms in a patient. These methods also inhibit the aggregation of bacteria that form biofilms in the airways. The methods include administering to a subject that has or is at risk of developing biofilms a compound or formulation that inhibits the formation or polymerization of actin microfilaments or depolymerizes actin microfilaments at or proximal to the site of biofilm formation. Such a compound can be administered in combination with a compound or formulation that inhibits the accumulation or activity of cells that are likely to undergo necrosis at or proximal to the site of biofilm formation (i.e., neutrophils). The methods and compositions can further include the use of anti-DNA and/or anti-mucin compounds, as well as other therapeutic compounds and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)from U.S. Provisional Application Ser. No. 60/599,495, filed Aug. 6,2004. The entire disclosure of U.S. Provisional Application Ser. No.60/599,495 is incorporated herein by reference in its entirety.

Government Support

This invention was supported, in part, using funds provided by NIH GrantNos. HL061407 and HL068743, each awarded by the National Institutes ofHealth, and using funds provided by NIAID Grant No. A115950, awarded bythe National Institute of Allergy and Infectious Diseases. Thegovernment has certain rights to this invention.

FIELD OF THE INVENTION

The present invention generally relates to methods and compositions forthe inhibition of biofilm formation or reduction of existing ordeveloping biofilms in a patient.

BACKGROUND OF THE INVENTION

Cystic fibrosis (CF) lung disease features persistent neutrophilaccumulation to the airways from the time of infancy (25). In theabsence of detectable infection or endotoxin, bronchioalveolar lavagestudies have recovered neutrophils ranging from 10⁴ to 10⁶ per ml fromthe airways of CF children (25, 29, 30). These children are frequentlyexposed to environmental strains of P. aeruginosa, but early infectionscan be transient, or be eradicated by aggressive antibiotic treatmentand an exuberant host defense (6, 17, 35). Initial success ineradicating P. aeruginosa acquired from environmental sources likelyoccurs due to a low density of organisms, a lack of antibioticresistance, and a generally nonmucoid phenotype. Eventually, persistentP. aeruginosa infection appears inevitable, and by adulthood, 80% of CFpatients are chronically infected (16).

Factors that allow P. aeruginosa to become persistent are of particularinterest, as chronic P. aeruginosa infection is clearly associated withincreased morbidity and mortality in CF patients (13, 32, 37). Thepersistent P. aeruginosa infection is associated with numerousphenotypic and genetic changes by the bacteria within the CF airway (11,14, 41) including the formation of biofilms (1, 11, 40). Bacterialbiofilms are surface-attached communities of cells encased within aself-produced extracellular polysaccharide matrix. Biofilm developmentproceeds through a series of programmed steps including initial surfaceattachment, formation of three-dimensional microcolonies, and finallythe development of a ‘mature’ biofilm (26). The detection of a specificpattern of quorum-sensing signaling molecules in the CF airway suggeststhat P. aeruginosa in the CF airway exists primarily in the biofilm form(40), and this conclusion is supported by the inability of antibioticsand host defense mechanisms to eradicate the infection (1, 11, 40).

Despite some promising advances, correction of CF by gene therapy is notyet attainable. Currently, antibiotic regimens coupled with drugs thatfacilitate the clearance of purulent airway secretions remain themainstay treatments for progressive airway disease. Inhalation ofpurified rhDNase (Pulmozyme; Genentech, USA), which digestsextracellular DNA present in the CF airway, is widely used as arespiratory decongestant. Such treatment is clinically effective fordiminishing sputum viscosity and stabilizing the forced expiratoryvolume (FEV) (Fuchs et al., N Engl J Med 331:637-642, 1994).

In addition to CF, a variety of other medical conditions and treatmentscan cause the undesirable development of biofilms. For example, avariety of microbial infections can be characterized by biofilmformation, including, but not limited to, infectious kidney stones,cystitis, catheter-related infection (kidney, vascular, peritoneal),medical device-related infections, prostatitis, dental caries, chronicotitis media, bronchiectasis, bacterial endocarditis, Legionnaire'sdisease, orthopedic implant infection, osteomyelitis, wounds, acne, andbiliary stents. Therefore, there is a need in the art for improvedtherapeutic approaches for the inhibition of biofilm formation and/orthe reduction or elimination of biofilms, which will be useful for thetreatment of conditions such as cystic fibrosis, as well as otherdiseases and conditions that are associated with the formation ofmicrobial biofilms.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a method to inhibitbiofilm formation or reduce biofilms in a subject. The method includesthe step of administering to a subject that has or is at risk ofdeveloping biofilms, a compound that inhibits the formation orpolymerization of actin microfilaments or depolymerizes actinmicrofilaments at or proximal to the site of biofilm formation or thesite of infection by a microorganism that forms biofilms. Typically, theactin microfilaments targeted by the present method are formed largelyfrom the content of a cell that undergoes necrosis at or proximal to thesite of biofilm formation or the site of infection by a microorganismthat forms biofilms. Such cells include, but are not limited to,neutrophils, airway epithelial cells or other epithelial cells,macrophages, monocytes, lymphocytes, eosinophils, and the infectiousmicrobe itself (e.g., P. aeruginosa).

Suitable compounds for use in this embodiment of the invention include,but are not limited to: cytochalasins, latrunculins, misakinolides,swinholides, myacolides, spinxolides, and scytophycins. Specificcompounds which are useful in the present invention include, but are notlimited to, cytochalasin B, cytochalasin D, latrunculin A, misakinolideA, swinholide A, myacolide B, spinxolide, scytophycin, domain 1 ofgelsolin, destrin or profilin

A further embodiment of the method of the present invention includes astep of administering to a subject a compound that inhibits accumulationof, inhibits necrosis of, or inhibits release of the cellular contentsof, cells that undergo necrosis, at or proximal to the site of biofilmformation or the site of infection by a microorganism that formsbiofilms. Such cells are described above. This step can be performed incombination with the administration of the anti-actin microfilamentcompound described above, or as an alternate method for inhibitingbiofilm formation or reducing biofilms in a subject. For example, thecompound preferably inhibits the adherence of, migration to, or thesensing or response to chemoattractants by neutrophils, or inhibits theactivity or release of a cytokine, chemokine or chemoattractant thatattracts or enhances neutrophil activity.

Suitable compounds for use in this embodiment of the invention include,but are not limited to: cytokine inhibitors, chemokine inhibitors,chemoattractant inhibitors, fluoroquinolones, Cox inhibitors, leukotienereceptor antagonists, leukotriene synthesis inhibitors, inhibitors ofthe p38 MAP kinase pathway, and glucocorticoids. More specifically,compounds that are useful in this embodiment of the invention include,but are not limited to: any inhibitor of eicosanoid synthesis andrelease, including any Cox-2 inhibitor; Cox-1 inhibitors; inhibitors ofsome certain prostaglandins (prostaglandin E(2); PGD(2)), inhibitors ofcertain leukotrienes (LTB₄); classes of antibiotics with known direct orindirect anti-inflammatory effects, including macrolides (e.g.azithromycin) and fluoroquinolones (e.g., levofloxacin; moxifloxacin;gatifloxacin); inhibitors of p38 MAP kinase; antagonists of growthfactors which regulate neutrophil release, including granulocytecolony-stimulating factor (G-CSF) (e.g., antibodies or antigen bindingfragments thereof, G-CSF antagonist variants or mimetics, drugs thatantagonize the function of G-CSF); antagonists of granulocyte-macrophagecolony-stimulating factor (GM-CSF); inhibitors of the function ofcytokines and chemokines, including antagonists of tumor necrosis factor(TNF), antagonists of interleukin-8 (IL-8); transforming growth factorbeta (TGF-beta); antibodies that block sites of neutrophil adhesion andthereby limit neutrophil accumulation to sites of inflammation,including anti-beta2 integrins (e.g., anti-CD11/CD18) and anti-ICAM-1;and neutrophil inhibitory material from other organisms, (e.g.,excretory-secretory (ES) material from the parasitic nematodeNippostrongylus brasiliensis). A preferred compound is ananti-inflammatory compound.

In any of the above-described embodiments, the method can furtherinclude administering to the subject an anti-DNA compound and/or ananti-mucin compound. The method can also include administering to thesubject a compound for treatment of a disease or condition associatedwith biofilm formation.

Preferably, the compound is administered when a disease or conditionassociated with biofilm formation is diagnosed or suspected. In oneaspect, the compound is administered prior to the treatment of thesubject with a process that may cause a biofilm to form in the patient.In one aspect, the compound is administered with a pharmaceuticallyacceptable carrier. In another aspect, the compound is administereddirectly to or proximal to the site of biofilm formation or potentialtherefore. In one aspect, the compound is administered to the lung orairways of the subject. In another aspect, the compound is applied to aprosthetic graft or administered to the subject receiving the graftprior to or during the implantation or utilization of the graft. Inanother aspect, the compound is applied to a catheter prior to or duringuse of the catheter by a subject. In another aspect, the compound isapplied to the site of a wound or to the wound dressing when the woundis treated. In yet another aspect, the compound is applied to a medicaldevice that contacts a subject tissue surface prior to or during use ofthe medical device by a subject.

The biofilms to be prevented, inhibited or treated may be caused by anycondition or disease. For example, the biofilm may form in connectionwith a disease or condition in an organ, tissue or body system (e.g.,lung, urinary tract, head and neck, vascular system, bone, skin,abdomen). Similarly, the biofilm may form on a surface of a tissue,organ or bodily part (e.g., lung, medium airways, ureter, urethra,bladder, prostate, mouth, ear, heart valve, vein, joint, bone, skin, andbile duct.). The biofilm may form in connection with a disease orcondition selected from: infectious kidney stones, cystitis,catheter-related infection (kidney, vascular, peritoneal), medicaldevice-related infections, prostatitis, dental caries, chronic otitismedia, cystic fibrosis, bronchiectasis, bacterial endocarditis,Legionnaire's disease, orthopedic implant infection, osteomyelitis,wounds, acne, and biliary stents.

The microorganism responsible for biofilm formation includes, but is notlimited to, Pseudomonas aeruginosa, Burkholderia multivorans,Streptococcus sanguis, Escherichia coli, and Streptococcus viridans.

Another embodiment of the present invention relates to a composition forinhibiting biofilm formation or reducing biofilms in a subject. Thecomposition can comprise any one, two, three, four, five, or morecompounds as described above. In one embodiment, the compositionincludes: (1) a first compound that inhibits the formation orpolymerization of actin microfilaments or depolymerizes actinmicrofilaments at or proximal to the site of biofilm formation; and (2)a second compound that is an anti-DNA compound. This composition canfurther include a compound that inhibits the accumulation of, necrosisof, or release of the cellular contents of, neutrophils at or proximalto the site of biofilm formation, and a carrier suitable for applicationto the site of biofilm formation. In another aspect, the composition caninclude: (1) a first compound that inhibits the formation orpolymerization of actin microfilaments or depolymerizes actinmicrofilaments at or proximal to the site of biofilm formation; and (2)a second compound that inhibits the accumulation of, necrosis of, orrelease of the cellular contents of, neutrophils at or proximal to thesite of biofilm formation, and a carrier suitable for application to thesite of biofilm formation.

Yet another embodiment of the present invention relates to a method toidentify a compound that inhibits necrotic cell-enhanced biofilmformation or reduces necrotic cell-enhanced biofilms in a subject. Themethod includes the steps of: (a) contacting a putative inhibitorycompound with a microbial culture in the presence and absence of apopulation of cells or a lysate thereof, wherein the microbial cultureforms biofilms and is in a planktonic state prior to contact with theputative inhibitory compound, and wherein the population of cellsundergoes necrosis in the presence of the microbial cells; and (b)measuring biofilm formation after contact with the putative regulatorycompound as compared to in the absence of the compound and as comparedto in the presence and absence of the population of cells or lysatethereof; wherein a decrease in biofilm formation in the presence of theputative regulatory compound and the presence of the population of cellsor lysate thereof, as compared to in the absence of the population ofcells or lysate thereof and as compared to in the absence of theputative regulatory compound, indicates that the putative regulatorycompound inhibits necrotic cell-enhanced biofilm formation or reducesnecrotic cell-enhanced biofilms. In one aspect, the population of cellsis a population of neutrophils. Other aspects of this method can beexpanded as described elsewhere herein.

BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION

FIG. 1A shows that the presence of neutrophils had little effect on thelong-term survival of P. aeruginosa.

FIG. 1B shows that the presence of neutrophils resulted in fewer viableP. aeruginosa in the planktonic state compared to P. aeruginosa in theabsence of neutrophils (mean±SD of CFU (n=4); *p<0.05).

FIG. 1C shows that neutrophils increased the number of viable P.aeruginosa in the biofilm state compared to P. aeruginosa alone whenmeasured simultaneously with P. aeruginosa in the planktonic state shownin FIG. 1B (mean±SD of CFU (n=4); *p<0.05).

FIG. 1D shows that neutrophils increased biofilm density (assayed by CVstaining) compared to P. aeruginosa alone by 4 hrs (mean±SD of O.D.measurements (n=21). *p<0.05).

FIG. 1E shows that exopolysaccharide staining of biofilm densitydemonstrated that the presence of neutrophils resulted in a greaterquantity of biofilm compared to P. aeruginosa in the absence ofneutrophils by 4 hrs (mean±SD of O.D. measurements (n=21). *p<0.05).

FIG. 2A shows that the presence of lysed neutrophils resulted in agreater quantity of biofilm compared to P. aeruginosa in the absence ofneutrophils by 4 hrs.

FIG. 2B shows that by 72 hrs, lysed neutrophils added at 0, 24, and 48hrs (hatched bar) achieved 92% of the biofilm development seen withviable neutrophils added at 0, 24, and 48 hrs (solid bar).

FIG. 2C shows that the presence of lysed neutrophils resulted in agreater quantity of biofilm compared to P. aeruginosa in the absence ofneutrophils by 4 hrs.

FIG. 2D shows that by 72 hrs, lysed neutrophils added at 0, 24, and 48hrs (hatched bar) achieved 94% of the biofilm development seen withviable neutrophils added at 0, 24, and 48 hrs (solid bar).

FIG. 3A shows that isolated neutrophil granule proteins failed toenhance the density of biofilm formation.

FIG. 3B shows that actin and DNA enhance P. aeruginosa biofilmdevelopment.

FIG. 3C shows the loss of biofilm enhancement by disruption of DNA andactin.

FIG. 4 shows actin binding by P. aeruginosa.

FIG. 5 shows that deletion of genes encoding the quorum-sensing signalsrhl (ΔrhlR), las (ΔlasR) or both (ΔrhlR/lasR) resulted in little changein biofilm development in the absence of neutrophils, and that thesemutant strains did not respond to neutrophils by developing a thickerbiofilm.

FIG. 6 shows that ΔpvdS strains formed biofilms equivalent to PA01 inthe presence of neutrophils and that a ΔtatC mutant also forms biofilmspoorly and was not effected by the presence of neutrophils.

FIG. 7 shows that the ΔmucA mutant or the mucoid CF strain FDR1demonstrate decreased biofilm development relative to PA01, but in thepresence of neutrophils, this phenotype was no longer observed.

FIG. 8 show the index of neutrophils-enhancement of mutant strains,where the density of the biofilm in the presence of neutrophils isplotted as a fold-increase of the biofilm density relative to the strainin the absence of neutrophils.

FIG. 9A shows that the deletion of genes encoding the quorum-sensingsignals rhl (ΔrhlR), las (ΔlasR) or both (ΔrhlR/lasR) resulted indecreased binding to F-actin, while deletion of genes encoding foralginate results in increased F-actin binding.

FIG. 9B shows that the extent of F-actin binding by PA01 and isogenicmutants significantly correlated with the neutrophil-induced fold-changeof biofilm growth.

FIG. 10 shows the effect of mucin on P. aeruginosa biofilm development.

FIG. 11 shows the effect of neutrophils on Bcc biofilm formation.

DETAILED DESCRIPTION OF INVENTION

The present invention generally relates to a composition and method forthe inhibition of biofilm formation or reduction of existing ordeveloping biofilms in a patient. The methods of the present inventioncan also inhibit the aggregation of bacteria that form biofilms in theairways. In one embodiment, the method comprises administering to apatient that has or is at risk of developing biofilms a compound orformulation that inhibits the formation or polymerization of actinmicrofilaments or depolymerizes actin microfilaments at or proximal tothe site of biofilm formation. In another, or additional, aspect, themethod comprises administering to a patient that has or is at risk ofdeveloping biofilms a compound or formulation that inhibits (reduces,decreases, prevents) neutrophil accumulation or activity at or proximalto the site of biofilm formation. These aspects of the invention arebased on the inventors' discovery that the presence of neutrophils atthe site of bacterial infection enhances initial bacterial biofilmdevelopment in patients with cystic fibrosis through the formation ofpolymers of actin microfilaments and DNA from necrotic neutrophils.Therefore, inhibition of the neutrophil accumulation and therefore thesubsequent polymerization of actin microfilaments and DNA at the site ofinfection will inhibit the formation and establishment of biofilms,providing a significant benefit to a patient. In addition, thisdiscovery can be extended to other diseases and conditions associatedwith biofilm formation and particularly where neutrophils are involvedin an inflammatory response to the disease or condition, and moreparticularly, when neutrophil association with an inflammatory processis chronic or prolonged.

Persistent neutrophil accumulation and necrosis in the CF airwaysresults in sputum highly-enriched with DNA, actin, and granule proteins,which are all clearly implicated in the pathogenesis of CF lung disease(2, 25, 28, 33, 36, 38). Based on the concept that early CF lung diseasefeatures low numbers of planktonic, environmental strains of P.aeruginosa entering a neutrophil-rich airway (6), the present inventorstested the effect of neutrophils on the earliest stages of P. aeruginosabiofilm formation using a concentration of neutrophils compatible withthe quantity of cells present in the airways of CF children prior topersistent P. aeruginosa infection, and concentrations of P. aeruginosaconsistent with early infection (7, 29).

The present inventors have found that neutrophils enhance initial P.aeruginosa biofilm development through formation of a biological matrixcomprised of actin and DNA polymers. These polymers are present in CFsputum, and disruption of the polymers dispersed associated P.aeruginosa and reduced biofilm development. Specifically, the biofilmenhancement coincides with a significant reduction of P. aeruginosa inthe planktonic phase, resulting in little decrease in the overall numberof viable bacteria after the first 4 hours of incubation. The mechanismof neutrophil biofilm enhancement was identified as being polymerscomprised of actin and DNA. The bacteria bind to F-actin, and disruptionof the polymers with DNase results in dispersion of the bacteria and areduction in biofilm development. The presence of these actin/DNApolymers, with co-localization of P. aeruginosa, was confirmed in bothneutrophil lysates and CF sputum. The introduction of additionalneutrophils after 24 and 48 hours further enhanced P. aeruginosa biofilmdevelopment, while exposure to fewer neutrophils resulted in a lesserdegree of biofilm enhancement (data not shown).

These findings demonstrate a potential maladaptation of the primaryinnate response, as cellular components from necrotic neutrophils canserve as a biological matrix to facilitate P. aeruginosa biofilmformation when eradication of infection fails. P. aeruginosa biofilmformation in the CF airways appears to occur in the context of stagnantmucous plugs which are lodged in the airway lumen, and are largelycomposed of dead and dying neutrophils (45, 46). The short lifespan ofthe neutrophil in the present inventors' study is consistent withneutrophil survival in the bloodstream and in other in vitro systemswhere typical survival is 6 to 18 hrs (12, 27). When neutrophils and P.aeruginosa are combined in vitro, neutrophil killing of planktonic P.aeruginosa is maximal at about 50 min (20), but subsequent necrosis ofthe leukocyte occurs rapidly, and over time the ability of the remainingviable neutrophils to ingest and kill P. aeruginosa is overshadowed bybacterial multiplication. In the presence of infection orpro-inflammatory stimuli, apoptosis is prevented or delayed, and cellsmay be viable up to 36 hours (12, 27). The concentration of neutrophils(10⁷/ml) used in this analysis was based on BAL sampling of the airwaysof CF infants prior to persistent P. aeruginosa infection whereneutrophil recovery ranged from 10⁴-10⁶ per ml (25, 29, 30), with anestimated recovery rate of approximately 1-2% (10). An even broaderrange of quantity of P. aeruginosa has been isolated from CF childrenduring early infection (7,29), and the inventors selected 10⁶ cfu/ml asa representative concentration.

In clinical settings that do not feature massive accumulation ofneutrophils, various host products have been found in association withbacterial biofilms. Heterogeneous salivary films contain secretory IgAand α-amylase, which represent a binding sites for Streptococcus sanguisin the formation of dental plaques (19). Nearly all types of in-dwellingmedical devices can become coated with host proteins, electrolytes andorganic materials which appear to contribute to the presence ofpersistent infection (8). Uropathogenic strains of E. coli can formorganized biofilm-like colonies within the cytoplasm of bladder cellsduring a phase of urinary tract infection (24), and clots comprised offibrin and platelets facilitate Streptococcus viridans survival inendocarditis (23). However, the potential of an immune cell, integral tohost defense, to increase formation of a bacterial biofilm has neverbeen reported.

Without being bound by theory, since virtually every eukaryotic cellcontains significant quantities of actin, it is possible that othernecrotic cells, in addition to neutrophils, could enhance P. aeruginosabiofilm development. Therefore, the present invention is not limited tothe inhibition of the accumulation of, necrosis of, or release of thecellular contents of, neutrophils, but rather is extended to othernecrotic cells that are present at a site of a microbial infection. Forexample, in a severe skin burn P. aeruginosa could conceivably utilizeactin and DNA from necrotic epithelial cells. Similarly, the presentinvention is not limited to the inhibition of biofilms associated withbacterial infection, as other microbes can also form biofilms (discussedbelow).

Recent reports have identified new mechanisms by which neutrophilssuccessfully kill bacteria. Neutrophils actively generate “neutrophilextracellular traps” (NETs) that bind to Staphylococcus aureus and otherbacteria (4). Viable neutrophils secrete NETs within minutes, whichappear to trap bacteria and augment killing by retaining themicroorganism in close proximity to a variety of anti-microbial granuleproteins (4). These delicate NETs are primarily composed of DNA, as wellas histones and granule proteins, but do not contain actin. In distinctcontrast, the polymers described herein are comprised of both actin andDNA, do not require the action of granule proteins, are a product of thenecrotic neutrophil, and increase the number of surviving P. aeruginosa(see FIG. 1A) over a period of days. Fragmentation of the NETs by DNaseincreased bacterial survival, while disruption of the actin-DNA polymersby DNase reduced biofilm formation. Thus construction of NETs representsan elegant mechanism of successful bacterial killing by the liveneutrophil, while actin-DNA enhancement of biofilm formation mayrepresent a maladaptive response to years of relentless accumulation andneutrophil death in the CF airway.

Although the neutrophil contains a number of proteins with significantanti-microbial potential, it appears that successful bacterial killingby granule proteins is highly dependent on the extracellular milieu.Recently, purified lactoferrin, a major component of the secondaryneutrophil granules, was found to prevent P. aeruginosa biofilmdevelopment (39). Although lactoferrin is relatively abundant in CFsputum, it is only one of at least 50 proteins contained withinneutrophil granules (3), and its inhibitory effect was not evident whenthe total content of neutrophil granules were combined with P.aeruginosa (FIG. 3A). It is likely that during Pseudomonas-inducedneutrophil necrosis, lactoferrin (and other potential beneficialproteins) are degraded by neutrophil- and Pseudomonas-derived proteases(5, 44).

The unique environment of the CF airway exerts selective pressures,which can result in profound genetic alterations within the bacteria. P.aeruginosa strains isolated at the time of initial infection resembleenvironmental strains, which are motile, nonmucoid, lack antibioticresistance, and have a smooth-type penta-acylated LPS. After years ofinfection, “CF-strains” of P. aeruginosa emerge, with an extensive arrayof altered phenotypes (6), including a mucoid, nonmotile phenotype,extensive resistance to antibiotics, and a rough-type,arabinomannan-modified, hexa/hepta-acylated LPS (11, 14). The PA01strain used in the studies described herein clearly resembles theenvironmental strains of early infection. Without being bound by theory,the present inventors' believe it is of importance that the enhancementof biofilm formation described here is achieved with a non-mucoid,non-CF strain of P. aeruginosa, as this may represent a mechanism thatallows environmental strains to initially persist in the CF airway. Oncepresent in the biofilm form, environmental P. aeruginosa strains wouldhave the opportunity to adapt to the intense inflammatory conditions andantibiotic treatment over decades without eradication.

Therefore, one embodiment of the present invention comprisesadministering to the patient, either directly or by application to acarrier, implant, catheter, medical device, or tissue or wound dressing:a compound that inhibits the formation or polymerization of actinmicrofilaments or depolymerizes actin microfilaments, at or proximal tothe site of biofilm formation. The method can further comprise, oralternatively include, a compound that inhibits the accumulation of,necrosis of, and/or release of cellular content of cells that undergonecrosis, at or proximal to the site of bacterial infection and/orbiofilm formation. The method can further comprise, in combination withone or both of the compounds above, the administration of an anti-DNAcompound and/or an anti-mucin compound and/or another compound that isuseful for the prevention and/or treatment of a disease or condition inthe patient, or that is useful in connection with a procedure beingperformed on the patient.

Preferably, the cells that undergo necrosis and are targeted by themethod of the invention are neutrophils, although other types of cellsthat can undergo necrosis at a site of microbial infection are alsoincluded in the invention. For example, such other cells include, butare not limited to, airway epithelial cells or other epithelial cells,macrophages, monocytes, lymphocytes, eosinophils, and the infectiousmicrobe itself (e.g., P. aeruginosa).

A biofilm is generally defined herein as a community of microorganismsattached to a solid surface. A biofilm community can include bacteria,fungi, yeasts, protozoa, and other microorganisms. More specifically, abiofilm is a surface-attached community of microbial cells encasedwithin a self-produced extracellular polysaccharide matrix that exhibitsproperties different from the planktonic microbial counterparts.Biofilms that are commonly found associated with human tissue and organsurfaces are frequently bacterial biofilms. In cystic fibrosis, by wayof example, both Pseudomonas aeruginosa and Burkholderia multivoransinfect and form biofilms in the lungs of patients having the disease.Other examples of microorganisms that form biofilms in tissues or onmedical devices or dressings include, but are not limited to:Streptococcus sanguis, E. coli, and Streptococcus viridans.

The method of the present invention can be used to treat any patient(subject, individual, animal) that has, is developing (biofilm formationis clinically evident or detectable to the skilled artisan, but has notyet fully formed), or is at risk of developing (no biofilm formation isyet detectable to the clinician or skilled artisan, but the subject isknown to be at risk of developing a biofilm due to disease or thepending performance of a treatment, such as a graft implantation). Theterm “patient” typically refers to a subject that is to be treated or isbeing treated by a clinician (doctor, nurse, or other medicalpractitioner) for a disease, condition, procedure, or routineexamination (i.e., the patient need not be ill or otherwise sufferingfrom any disease or condition to be treated). However, as used herein,the terms “patient”, “subject”, “individual” and “animal” can begenerally be used interchangeably with reference to the subject to whicha compound of the invention is to be administered.

Microbial biofilms can form in and on a variety of tissues as well as onor in a variety of devices and materials that may be used during thetreatment of a subject for a particular disease or condition. Forexample, the method of the present invention can be used to prevent orreduced biofilm formation, or to reduce existing or developing biofilmsthat may form in connection with a disease or condition in an organ,tissue or body system including, but not limited to, lung, urinarytract, head and neck, vascular system, bone, skin, abdomen. Biofilms mayalso form on the surface of a tissue, organ or bodily part including,but not limited to, lung, medium airways, ureter, urethra, bladder,prostate, mouth, ear, heart valve, vein, joint, bone, skin, and bileduct. Biofilms may form in connection with a disease or conditionincluding, but not limited to: infectious kidney stones, cystitis,catheter-related infection (kidney, vascular, peritoneal), medicaldevice-related infections, prostatitis, dental caries, chronic otitismedia, cystic fibrosis, bronchiectasis, bacterial endocarditis,Legionnaire's disease, orthopedic implant infection, osteomyelitis,wounds, acne, and biliary stents. All of these scenarios are encompassedby the present invention.

Preferably, the compound is administered to a subject (patient,individual, animal) prior to the development of a biofilm, or at theearliest time that biofilm development is suspected or detected. Withoutbeing bound by theory, the present inventors believe that the method ofthe present invention will be particularly effective when used as apreventative or early stage inhibition of biofilm formation. Forexample, the method of the invention can be used when a patient issuspected to have or be developing a disease or condition associate withthe formation of biofilms, where the method is used when the diagnosismade or early treatment is performed (e.g., prior to the establishmentof biofilms in the patient, although there may be detectable evidence ofbiofilm formation). A young patient diagnosed with cystic fibrosis, forexample, may develop biofilms after several years of the disease, butduring the earlier diagnosis and treatment stages, the method of theinvention may prevent or reduce the formation of the biofilms as thedisease advances in the patient. As another example, the method of thepresent invention may be applied to a prosthetic graft or used in apatient receiving the graft prior to or during the implantation orutilization of the graft. Similarly, the method of the present inventioncan be used when prior to or during use of a catheter by a patient, byapplying the compound to the catheter and/or on the tissue contacting ornear the catheter. The compound could also be applied to the site of awound or to the wound dressing when the wound is initially andsubsequently treated, or the compound can be applied to a medical devicethat contacts a patient tissue surface prior to or during use of themedical device by a patient.

For the inhibition of the formation or polymerization of actinmicrofilaments (or depolymerization of the actin microfilaments),compounds preferably inhibit F-actin, which is the microfilament form ofactin, and can also be referred to herein as “anti-actin” compounds. Avariety of compounds that affect the polymerization and depolymerizationof actin filaments are well known in the art. A detailed description ofvarious classes of such compounds, as well as specific compounds andtheir known actions on actin is provided in Meijer et al., 2003,Progress in Cell Cycle Research 5:511-525, which is incorporated hereinby reference in its entirety. Classes of compounds that can be used inthe present invention include, but are not limited to, cytochalasins,latrunculins, misakinolides, swinholides, myacolides, spinxolides, andscytophycins. Specific compounds which are useful in aproduct/composition/formulation of the present invention include, butare not limited to, cytochalasin B, cytochalasin D, latrunculin A,misakinolide A, swinholide A, myacolide B, spinxolide, scytophycin,domain 1 of gelsolin, destrin or profilin. Other suitable anti-actincompounds will be known to those of skill in the art or can beidentified using standard actin polymerization assays (e.g., see Meijeret al., supra) and such compounds are encompassed for use in the presentinvention.

For the inhibition of neutrophil accumulation, necrosis and/or releaseof cellular content, or for the inhibition of many other necrotic celltypes targeted by the invention (cells that can undergo necrosis at thesite of a microbial infection), any anti-inflammatory compound or anycompound that interferes with a neutrophil's (by way of example) abilityto adhere to or near a site of infection by biofilm-associated microbe,to migrate to such site, or to sense or respond to chemoattractants ator near such site (or that would result in migration of the neutrophilto such site), is encompassed by the present invention. For example,such compounds can inhibit or reduce the release or biological activityof chemoattractants, cytokines, or chemokines at or near (proximal to)the site of infection that would otherwise attract a neutrophil, causeit to migrate to the site of infection, or allow or enhance neutrophiladherence at or near the site of infection. Administration of suchanti-inflammatory/anti-neutrophil compounds early in the disease processthat is associated with biofilm formation is believed to be an importantaspect of the invention. Therefore, anti-inflammatories/anti-neutrophilcompounds would be administered upon the initial diagnosis of thedisease or condition that is associated with biofilm formation, andpreferably prior to a significant formation of biofilms in the patient.

Such compounds are well known in the art and include, but are notlimited to, cytokine inhibitors, chemokine inhibitors, chemoattractantinhibitors, fluoroquinolones, Cox inhibitors, leukotiene receptorantagonists, leukotriene synthesis inhibitors, inhibitors of the p38 MAPkinase pathway, and glucocorticoids. More specifically, compounds thatare useful in this embodiment of the invention include, but are notlimited to: any inhibitor of eicosanoid synthesis and release, includingany Cox-2 inhibitor; Cox-1 inhibitors; inhibitors of some certainprostaglandins (prostaglandin E(2); PGD(2)), inhibitors of certainleukotrienes (LTB₄); classes of antibiotics with known direct orindirect anti-inflammatory effects, including macrolides (e.g.azithromycin) and fluoroquinolones (e.g., levofloxacin; moxifloxacin;gatifloxacin); inhibitors of p38 MAP kinase; antagonists of growthfactors which regulate neutrophil release, including granulocytecolony-stimulating factor (G-CSF) (e.g., antibodies or antigen bindingfragments thereof, G-CSF antagonist variants or mimetics, drugs thatantagonize the function of G-CSF); antagonists of granulocyte-macrophagecolony-stimulating factor (GM-CSF); inhibitors of the function ofcytokines and chemokines, including antagonists of tumor necrosis factor(TNF), antagonists of interleukin-8 (IL-8); transforming growth factorbeta (TGF-beta); antibodies that block sites of neutrophil adhesion andthereby limit neutrophil accumulation to sites of inflammation,including anti-beta2 integrins (e.g., anti-CD11/CD18) and anti-ICAM-1;and neutrophil inhibitory material from other organisms, (e.g.,excretory-secretory (ES) material from the parasitic nematodeNippostrongylus brasiliensis).

In one embodiment, a product, composition or formulation of the presentinvention also includes an anti-DNA compound. According to the presentinvention, an anti-DNA compound is any compound that causes thedestabilization or degradation of DNA. Such compounds are known in theart and include, but are not limited to, nucleases, hydroxyl radicalgenerating compounds, and the like. For example, DNase I or rhDNase(Pulmozyme; Genentech, USA) is a well-known anti-DNA compound that isuseful in the present invention. Compounds suitable for the degradationof DNA will be known to those of skill in the art and all areencompassed by the present invention.

In another embodiment, a product, composition or formulation of thepresent invention also includes an anti-mucin compound. Mucins are afamily of large, heavily glycosylated proteins. Some mucins are membranebound due to the presence of a hydrophobic membrane-spanning domain thatfavors retention in the plasma membrane, but many mucins are secreted onmucosal surfaces and in saliva. Anti-mucin compounds include anycompound that causes the destabilization or degradation of mucin, orinhibits the interaction of mucin with other compounds or molecules.Such compounds include, but are not limited to, antibodies and antigenbinding fragments thereof that bind to mucin, sulphatases, glycosidases,and proteases.

In another embodiment, a product, composition or formulation of thepresent invention also includes one or more compounds that are usefulfor treating a particular disease or condition that is associated withbiofilm formation. For example, when the patient has or is suspected ofhaving cystic fibrosis, the anti-actin compound and/oranti-inflammatory/anti-neutrophil compound can be used in conjunctionwith other drugs or therapeutic compounds that are conventionally usedto treat cystic fibrosis. As another example, when the patient has awound, the anti-actin compound and/or anti-inflammatory/anti-neutrophilcompound can be applied to the wound dressing, along with othercompounds, such as anti-microbial compounds, or administeredconcurrently with other such compounds by a different route. As yetanother example, patients receiving a prosthetic graft may be receivinganti-rejection drugs, anti-microbials, or growth factors to enhance theestablishment of the graft or growth of appropriate tissue at the graftsite, and the anti-actin compound and/oranti-inflammatory/anti-neutrophil compound of the invention can beadministered in connection with such treatments.

According to the present invention, the present invention can use anyone, two, three, four, or more compounds from any class listed above,including any combination of the compounds. For example, in a preferredembodiment, the method uses both an anti-actin microfilament compoundand an anti-DNA compound and/or an anti-mucin compound. Alternatively,the method uses both an anti-actin microfilament compound and ananti-neutrophil (or other necrotic cell) compound. In another aspect,the method uses an anti-neutrophil (or other necrotic cell) compound andan anti-DNA compound and/or an anti-mucin compound. In furtherembodiments, additional compounds that are useful for the treatment of aparticular condition or disease in the patient to be treated can beincluded.

According to the present invention, an “antagonist” or an“anti”-compound or agent (e.g., an anti-actin microfilament compound,an-anti-neutrophil compound, an anti-DNA compound or an anti-mucincompound) refers to any compound which inhibits (e.g., antagonizes,reduces, decreases, blocks, reverses, or alters) the effect of a givenprotein or compound. More particularly, an antagonist is capable ofacting in a manner relative to the given protein's or compound'sactivity, such that the biological activity of the given protein orcompound, is decreased or blocked in a manner that is antagonistic(e.g., against, a reversal of, contrary to) to the natural action of thegiven protein or compound. Antagonists can include, but are not limitedto, an antibody or antigen binding fragment thereof, a protein, peptide,nucleic acid (including ribozymes and antisense), or a product ofdrug/compound/peptide design or selection that provides the antagonisticeffect.

Antagonists useful in the present invention also include compounds thatare products of rational drug design, natural products, and compoundshaving partially defined regulatory properties. A regulatory agent,including an antagonist of a given protein, can be a protein-basedcompound, a carbohydrate-based compound, a lipid-based compound, anucleic acid-based compound, a natural organic compound, a syntheticallyderived organic compound, or an antibody, or fragments thereof. In oneembodiment, such regulatory agents of the present invention includedrugs, including peptides, oligonucleotides, carbohydrates and/orsynthetic organic molecules which regulate the production and/orfunction of one or more proteins in the alternative complement pathway.Such an agent can be obtained, for example, from molecular diversitystrategies (a combination of related strategies allowing the rapidconstruction of large, chemically diverse molecule libraries), librariesof natural or synthetic compounds, in particular from chemical orcombinatorial libraries (i.e., libraries of compounds that differ insequence or size but that have the same building blocks) or by rationaldrug design. See for example, Maulik et al., 1997, MolecularBiotechnology: Therapeutic Applications and Strategies, Wiley-Liss,Inc., which is incorporated herein by reference in its entirety.

In a molecular diversity strategy, large compound libraries aresynthesized, for example, from peptides, oligonucleotides, carbohydratesand/or synthetic organic molecules, using biological, enzymatic and/orchemical approaches. The critical parameters in developing a moleculardiversity strategy include subunit diversity, molecular size, andlibrary diversity. The general goal of screening such libraries is toutilize sequential application of combinatorial selection to obtainhigh-affinity ligands against a desired target, and then optimize thelead molecules by either random or directed design strategies. Methodsof molecular diversity are described in detail in Maulik, et al., supra.

In a rational drug design procedure, the three-dimensional structure ofa regulatory compound can be analyzed by, for example, nuclear magneticresonance (NMR) or X-ray crystallography. This three-dimensionalstructure can then be used to predict structures of potential compounds,such as potential regulatory agents by, for example, computer modeling.The predicted compound structure can be used to optimize lead compoundsderived, for example, by molecular diversity methods. In addition, thepredicted compound structure can be produced by, for example, chemicalsynthesis, recombinant DNA technology, or by isolating a mimetope from anatural source (e.g., plants, animals, bacteria and fungi).

Various other methods of structure-based drug design are disclosed inMaulik et al., 1997, supra. Maulik et al. disclose, for example, methodsof directed design, in which the user directs the process of creatingnovel molecules from a fragment library of appropriately selectedfragments; random design, in which the user uses a genetic or otheralgorithm to randomly mutate fragments and their combinations whilesimultaneously applying a selection criterion to evaluate the fitness ofcandidate ligands; and a grid-based approach in which the usercalculates the interaction energy between three dimensional receptorstructures and small fragment probes, followed by linking together offavorable probe sites.

An antibody or antigen binding fragment thereof useful in the presentinvention selectively binds to a protein and thereby blocks or inhibitsthe activity of the protein. According to the present invention, thephrase “selectively binds to” refers to the ability of an antibody,antigen binding fragment or binding partner to preferentially bind tospecified proteins. More specifically, the phrase “selectively binds”refers to the specific binding of one protein to another (e.g., anantibody, fragment thereof, or binding partner to an antigen), whereinthe level of binding, as measured by any standard assay (e.g., animmunoassay), is statistically significantly higher than the backgroundcontrol for the assay. For example, when performing an immunoassay,controls typically include a reaction well/tube that contain antibody orantigen binding fragment alone (i.e., in the absence of antigen),wherein an amount of reactivity (e.g., non-specific binding to the well)by the antibody or antigen binding fragment thereof in the absence ofthe antigen is considered to be background. Binding can be measuredusing a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, etc.

Isolated antibodies of the present invention can include serumcontaining such antibodies, or antibodies that have been purified tovarying degrees. Whole antibodies of the present invention can bepolyclonal or monoclonal. Alternatively, functional equivalents of wholeantibodies, such as antigen binding fragments in which one or moreantibody domains are truncated or absent (e.g., Fv, Fab, Fab′, or F(ab)₂fragments), as well as genetically-engineered antibodies or antigenbinding fragments thereof, including single chain antibodies orantibodies that can bind to more than one epitope (e.g., bi-specificantibodies), or antibodies that can bind to one or more differentantigens (e.g., bi- or multi-specific antibodies), may also be employedin the invention. A bi-specific (or multi-specific) antibody is capableof binding two (or more) antigens, as with a divalent (or multivalent)antibody, but in this case, the antigens are different antigens (i.e.,the antibody exhibits dual or greater specificity). A bi-specificantibody suitable for use in the present method includes an antibodyhaving: (a) a first portion (e.g., a first antigen binding portion)which binds to a given protein; and (b) a second portion which binds toa second protein.

The invention also extends to non-antibody polypeptides, sometimesreferred to as antigen binding partners or antigen binding polypeptides,that have been designed to bind selectively to and cause theneutralization or inhibition of a protein according to the presentinvention. Examples of the design of such polypeptides, which possess aprescribed ligand specificity are given in Beste et al. (Proc. Natl.Acad. Sci. 96:1898-1903, 1999), incorporated herein by reference in itsentirety.

An isolated nucleic acid molecule that is useful as an antagonistincludes, but is not limited to, an anti-sense nucleic acid molecule, aribozyme or siRNA. As used herein, an anti-sense nucleic acid moleculeis defined as an isolated nucleic acid molecule that reduces expressionof a protein by hybridizing under high stringency conditions to a geneencoding the protein. Such a nucleic acid molecule is sufficientlysimilar to the gene encoding the protein that the molecule is capable ofhybridizing under high stringency conditions to the coding orcomplementary strand of the gene or RNA encoding the natural protein.RNA interference (RNAi) is a process whereby double stranded RNA, and inmammalian systems, short interfering RNA (siRNA), is used to inhibit orsilence expression of complementary genes. In the target cell, siRNA areunwound and associate with an RNA induced silencing complex (RISC),which is then guided to the mRNA sequences that are complementary to thesiRNA, whereby the RISC cleaves the mRNA. A ribozyme is an RNA segmentthat functions by binding to the target RNA moiety and inactivate it bycleaving the phosphodiester backbone at a specific cutting site.

An isolated nucleic acid molecule is a nucleic acid molecule that hasbeen removed from its natural milieu (i.e., that has been subject tohuman manipulation) and can include DNA, RNA, or derivatives of eitherDNA or RNA. As such, “isolated” does not reflect the extent to which thenucleic acid molecule has been purified. An isolated nucleic acidmolecule can be isolated from its natural source or produced usingrecombinant DNA technology (e.g., polymerase chain reaction (PCR)amplification, cloning) or chemical synthesis.

As used herein, reference to hybridization conditions refers to standardhybridization conditions under which nucleic acid molecules are used toidentify similar nucleic acid molecules. Such standard conditions aredisclosed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al.,ibid., is incorporated by reference herein in its entirety (seespecifically, pages 9.31-9.62). In addition, formulae to calculate theappropriate hybridization and wash conditions to achieve hybridizationpermitting varying degrees of mismatch of nucleotides are disclosed, forexample, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkothet al., ibid., is incorporated by reference herein in its entirety.

In one embodiment of the invention, a method is provided to identifycompounds that are useful in the method of inhibiting the formation ofbiofilms that are enhanced by necrotic cells, such as neutrophils, orreducing existing or developing such biofilms. This method includes thesteps of: (a) contacting a putative inhibitory compound with a microbialculture in the presence and absence of a population of cells or a lysatethereof; and (b) measuring biofilm formation after contact with theputative regulatory compound as compared to in the absence of thecompound and as compared to in the presence and absence of thepopulation of cells or lysate thereof. A decrease in biofilm formationin the presence of the putative regulatory compound and the presence ofthe population of cells or lysate thereof, as compared to in the absenceof the population of cells or lysate thereof and as compared to in theabsence of the putative regulatory compound, indicates that the putativeregulatory compound inhibits necrotic cell-enhanced biofilm formation orreduces necrotic cell-enhanced biofilms. In this embodiment of theinvention, the microbial culture must be a microbial culture that canform biofilms, but the culture is provided in a planktonic state priorto contact with the putative inhibitory compound. In addition, thepopulation of cells used in this assay are selected because they canundergo necrosis in the presence of the microbial cells. For example, asshown in the examples, neutrophils will undergo necrosis in the presenceof a culture of P. aeruginosa. These are only exemplary microbe and cellcombinations that can be used. Other combinations will be apparent tothose of skill in the art. The assay of the invention is designed toidentify compounds that inhibit biofilm formation that is associatedwith or enhanced by the necrosis of cells at the site of infection orbiofilm formation.

Preferred microbial cells for use in this invention are any microbialcells that are capable of forming biofilms under some conditions, andparticularly, including in the presence of necrotic cells or thecomponents thereof (e.g., actin microfilaments or DNA) as describedherein. Preferably, formation of biofilms and aggregation of themicrobial cells is enhanced by the necrotic cells or components thereof.The microbial cells are not required to be of the same strain, species,genus, or even microbe, although this is preferred. Microbial cells thatform biofilms can include, but are not limited to, bacteria, fungi,yeasts, and protozoa, with bacteria being particularly preferred.Bacteria that are particularly useful in this method of the inventioninclude, but are not limited to, any of the previously described biofilmforming bacteria, such as P. aeruginosa, Burkholderia multivorans,Streptococcus sanguis, E. coli, and Streptococcus viridans. In oneembodiment, particular strains or mutants of a microbial cell can beused in the assay to identify compounds that impact necroticcell-enhanced biofilm formation that may be relevant to a particularstrain of microbial cell carried by a specific patient or subset ofpatients, or to focus the identification of the inhibitor on aparticular characteristic or expression of a particular gene or proteinin the microbial cell that affects necrotic cell-enhanced biofilmformation. In one embodiment, the cells can be labeled with a detectablelabel (e.g., green fluorescent protein).

Preferred populations of cells (or the lysates thereof) to be used inthe present invention include any cells that can undergo necrosis in thepresence of a microbial cell as described above. For example, such cellsinclude, but are not limited to, neutrophils, airway epithelial cells orother epithelial cells, macrophages, monocytes, lymphocytes,eosinophils, and the infectious microbe itself (e.g., P. aeruginosa).Cell lysates can be produced using any methods known to those of skillin the art, including any means of disrupting, permeabilizing orotherwise lysing of cell membranes to release the intracellularcontents. In one embodiment, the cells can be labeled with a detectablelabel.

As used herein, the term “test compound”, “putative inhibitory compound”or “putative regulatory compound” refers to compounds having an unknownor previously unappreciated regulatory activity in a particular process.As such, the term “identify” with regard to methods to identifycompounds is intended to include all compounds, the usefulness of whichas a regulatory compound for the purposes of regulating biofilmformation is determined by a method of the present invention.

The conditions under which a microbial cell are contacted with aputative regulatory compound according to the present invention, such asby mixing, plating, etc., are conditions in which the microbial cell isnot forming a biofilm (i.e., the microbial culture is in a planktonicstate) if essentially no regulatory compound is present. The conditionsunder which the population of cells that can undergo necrosis arecontacted with a putative regulatory compound are conditions under whichthe majority of cells in the population of cells are viable and notundergoing necrosis.

The present methods involve contacting cells and/or lysates with thecompound being tested for a sufficient time to allow for interaction ofthe compound with the microbial cells and/or the population of cells orcomponents in the lysate thereof. The period of contact with thecompound being tested can be varied depending on the result beingmeasured, and can be determined by one of skill in the art. As usedherein, the term “contact period” refers to the time period during whichcells are in contact with the compound being tested. The term“incubation period” refers to the entire time during which cells areallowed to grow prior to evaluation, and can be inclusive of the contactperiod. Thus, the incubation period includes all of the contact periodand may include a further time period during which the compound beingtested is not present but during which growth is continuing (in the caseof a cell based assay) prior to scoring.

The final step in the method is to measure biofilm formation or aparameter associated with biofilm formation in the presence and absenceof the putative regulatory compound and in the presence and absence ofthe population of cells. Since the present method is designed toidentify compounds that impact necrotic cell-enhanced biofilm formation(although the compound may also affect biofilm formation in the absenceof necrotic cells), a candidate compound is identified as useful if itinhibits biofilm formation to a greater degree (detectable, andpreferably, statistically significantly greater) in the presence of thenecrotic cells as compared to in the absence of the necrotic cells.Statistical analysis to determine differences between controls and testcultures can be performed using any methods known in the art, including,but not limited to, Student's t test or analysis of variance forcontinuous variables. Statistical significance is typically defined asp<0.05.

In another, or additional embodiment, one can detect the effect of theputative regulatory compound on the binding of the microbial cells toactin and/or DNA from the population of necrotic cells, or on theaggregation of microbial cells in the presence of the population ofnecrotic cells.

Methods of evaluating biofilm formation are well known in the art andare described in the Examples. For example, confocal microscopy,microscopy, and static biofilm assays. Methods of measuring actin andDNA binding are also well known in the art and are described in theExamples.

Agonists and antagonists identified by the above methods or any othersuitable method are useful in the therapeutic or biofilm-inhibitionmethods as described herein.

Compounds useful in the present invention are typically provided in theform of a composition (formulation). In one embodiment of the invention,a pharmaceutical composition or formulation is prepared from aneffective amount of a compound of the invention and apharmaceutically-acceptable carrier. Pharmaceutically-acceptablecarriers are well known to those with skill in the art. According to thepresent invention, a “pharmaceutically acceptable carrier” includespharmaceutically acceptable excipients and/or pharmaceuticallyacceptable delivery vehicles, which are suitable for use in theadministration of a formulation or composition to a suitable in vivosite. A suitable in vivo site is preferably any site wherein biofilmshave formed, are forming, or may form. Preferred pharmaceuticallyacceptable carriers are capable of maintaining a compound used in aformulation of the invention in a form that, upon arrival of thecompound at the target site in a patient, the compound is capable ofacting, preferably resulting in a therapeutic benefit to the patient.

One type of pharmaceutically acceptable carrier includes a controlledrelease formulation that is capable of slowly releasing a composition ofthe present invention into a patient. Suitable controlled releasevehicles include, but are not limited to, biocompatible polymers, otherpolymeric matrices, capsules, microcapsules, microparticles, boluspreparations, osmotic pumps, diffusion devices, liposomes, lipospheres,and transdermal delivery systems. Other suitable carriers include anycarrier that can be bound to or incorporated with the compound thatextends that half-life of the compound to be delivered. A carrier can bemodified to target to a particular site in a patient, thereby targetingand making use of a compound at that site.

In one embodiment, a compound useful in the present method isadministered in a formulation suitable for aerosol delivery. Carriersthat are particularly useful for aerosol delivery according to thepresent invention include, but are not limited to: dry, dispersiblepowders; small capsules (e.g., microcapsules or microparticles);liposomes; and nebulized sprays. Dry, dispersible powders suitable foraerosolized delivery of compounds are described in detail in U.S. Pat.No. 6,165,463, incorporated herein by reference in its entirety (Seealso products from Inhale Therapeutic Systems, Inc. and QuadrantTechnology). Suitable liposomes for use in aerosols include anyliposome, and particularly, any liposome that is sufficiently small tobe delivered by aerosol in the method of the invention. Microcapsulesand microparticles are known in the art. For example, AlliancePharmaceutical Corporation has a particle engineering technology, calledPulmoSphere, prepared by a proprietary spray-drying process and aredesigned to be both hollow and porous. A product by Ventolin consists ofmicronized albuterol (free base) particles suspended in a mixture ofCFC-based propellants. Proventil HFA contains micronized albuterolsulfate and a small percentage of an ethanol co-solvent to solubilizethe stabilizing oleic acid surfactant. Devices for delivery ofaerosolized formulations include, but are not limited to, pressurizedmetered dose inhalers (MDI), dry powder inhalers (DPI), and meteredsolution devices (MSI), and include devices that are nebulizers andinhalers.

In another embodiment, a compound useful in the present method isadministered in a formulation suitable for topical delivery. Suchformulations include any lotion, excipient, cream, gel, or other topicalcarrier suitable for topical administration.

For injection, the compounds of the invention can be formulated inappropriate aqueous solutions, such as physiologically compatiblebuffers such as Hanks's solution, Ringer's solution, or physiologicalsaline buffer.

For transmucosal and transcutaneous administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated.

The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In accordance with the present invention, determination of acceptableprotocols to administer a compound (product, agent, composition,formulation), including the route of administration and the effectiveamount of a compound to be administered to a patient, can beaccomplished by those skilled in the art. A compound of the presentinvention can be administered in vivo or ex vivo. Suitable in vivoroutes of administration can include, but are not limited to, oral,nasal, inhaled, topical, intratracheal, transdermal, rectal, andparenteral routes. Preferred parenteral routes can include, but are notlimited to, subcutaneous, intradermal, intravenous, intramuscular, andintraperitoneal routes. Preferred topical routes include inhalation byaerosol (i.e., spraying) or topical surface administration to the skinof a patient, or to a dressing, device, catheter, prosthetic graft orother product to be placed into contact with a patient tissue surface.Preferably, a compound is administered by nasal, inhaled, intratracheal,topical, or systemic routes (e.g., intraperitoneal, intravenous). Exvivo refers to performing part of the administration step outside of thepatient.

Preferably, the compound is administered directly to or proximal to thesite of biofilm formation or potential therefore. For example, thebiofilm can be administered by surgical or clinical procedure directlyto the tissue, organ, bodily part, or to a material or device that is tobe (or anticipated to be) at or proximal to a site in the patient wherea biofilm may form, is likely to form, or will form. By way of example,in one aspect, the compound is administered to the lung or airways ofthe patient. In another aspect, the compound is applied to a prostheticgraft or administered to the patient receiving the graft prior to orduring the implantation or utilization of the graft. In another aspect,the compound is applied to a catheter prior to or during use of thecatheter by a patient. In yet another aspect, the compound is applied tothe site of a wound or to the wound dressing when the wound is treated.A compound may also be applied to a medical device that contacts apatient tissue surface prior to or during use of the medical device by apatient. Other types of administration or application of the compoundand method of the invention will be apparent to those of skill in theart given this discussion.

An effective amount is any amount of the compound that causes adetectable reduction in biofilm formation as compared to in the absenceof the compound, or reduces existing biofilms or developing biofilms ascompared to in the absence of the compound. A preferred single dose ofan agent, including proteins, small molecules and antibodies, for use ina method described herein, comprises between about 0.01microgram×kilograms⁻¹ and about 10 milligram×kilogram⁻¹ body weight ofan animal. A more preferred single dose of an agent comprises betweenabout 1 microgram×kilograms⁻¹ and about 10 milligram×kilogram⁻¹ bodyweight of an animal. An even more preferred single dose of an agentcomprises between about 5 microgram×kilogram⁻¹ and about 7milligram×kilogram⁻¹ body weight of an animal. An even more preferredsingle dose of an agent comprises between about 10 microgram×kilogram⁻¹and about 5 milligram×kilogram⁻¹ body weight of an animal. Aparticularly preferred single dose of an agent comprises between about0.1 milligram×kilogram⁻¹ and about 5 milligram×kilogram⁻¹ body weight ofan animal, if the an agent is delivered by aerosol. Another particularlypreferred single dose of an agent comprises between about 0.1microgram×kilogram⁻¹ and about 10 microgram×kilogram⁻¹ body weight of ananimal, if the agent is delivered parenterally.

In one embodiment, an appropriate single dose of a nucleic acid, whendelivered with a liposome carrier, is from about 0.1 μg to about 100 μgper kg body weight of the patient to which the complex is beingadministered. In another embodiment, an appropriate single dose is fromabout 1 μg to about 10 μg per kg body weight. In another embodiment, anappropriate single dose of nucleic acid:lipid complex is at least about0.1 μg of nucleic acid, more preferably at least about 1 μg of nucleicacid, even more preferably at least about 10 μg of nucleic acid, evenmore preferably at least about 50 μg of nucleic acid, and even morepreferably at least about 100 μg of nucleic acid.

One of skill in the art will be able to determine that the number ofdoses of a compound to be administered to an animal is dependent uponthe extent of the biofilm formation and the underlying condition ordisease of which biofilm formation is a symptom or a component, and theresponse of an individual patient to the treatment. In addition, theclinician will be able to determine the appropriate timing for deliveryof the compound in a manner effective to inhibit biofilm formation orreduce biofilms in the patient. Preferably, the compound is deliveredwithin between about 1 hour and 48 hours of the diagnosis orconfirmation by a clinician of the risk or likelihood of developingbiofilms or a condition or disease that is associated with thedevelopment of biofilms, or the as soon as an infection has beenidentified that would be likely to be associated with biofilms, or assoon thereafter as practical in order to inhibit biofilm formationbefore it develops or before it begins to have a deleterious effect onthe patient. When a medical device (graft, catheter, stent, wounddressing, prosthetic) is to be introduced into contact with a patienttissue surface, the compound is preferably administered prior to,concurrently with, or substantially immediately after the patient iscontacted with the device, graft or dressing. In one embodiment, thecompound is administered as soon as it is recognized (i.e., immediatelyor in a few hours or days) by the patient or clinician that the patientmay be at risk of or developing biofilms. Preferably, suchadministrations are given until the patient is no longer at risk ofdeveloping biofilms or at least until signs of biofilm inhibition orprevention occur. Preferably, the compound is administered within atleast 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, orany increment of 0.25 hours from 0.25 hours (15 minutes) to 72 hoursprior to or after the diagnosis, procedure or treatment of the patient.The compound can be administered subsequently, routinely, or as neededto prevent, control or reduce biofilm formation or reduce existing ordeveloping biofilms in the patient.

Typically, it is desirable to obtain a therapeutic benefit in a patient.A therapeutic benefit is not necessarily a cure for a particular diseaseor condition, but rather, preferably encompasses a result which caninclude alleviation of the disease or condition, elimination of thedisease or condition, reduction of a symptom associated with the diseaseor condition (e.g., biofilm formation), prevention or alleviation of asecondary disease or condition resulting from the occurrence of aprimary disease or condition, and/or prevention of the disease orcondition. A beneficial effect can easily be assessed by one of ordinaryskill in the art and/or by a trained clinician who is treating thepatient. The term, “disease” refers to any deviation from the normalhealth of the individual and includes a state when disease symptoms arepresent, as well as conditions in which a deviation (e.g., infection,gene mutation, genetic defect, etc.) has occurred, but symptoms are notyet manifested. As used herein, the phrase “protected from a disease”refers to reducing the symptoms of the disease; reducing the occurrenceof the disease, and/or reducing the severity of the disease. Protectinga patient can refer to the ability of a compound or composition of thepresent invention, when administered to a patient, to prevent acondition from occurring and/or to cure or to alleviate the symptoms ofthe disease or condition, signs or causes. As such, to protect a patientfrom a disease includes both preventing disease occurrence (prophylactictreatment) and treating a patient that has a disease (therapeutictreatment) to reduce the symptoms of the disease. More specifically,protecting a patient from biofilm formation can refer to preventing theformation or development of a biofilm and/or to reduce or eliminateexisting biofilms in the patient. To treat a patient refers to the actof applying the method of the invention to any suitable patient(subject, individual, animal).

In one embodiment of the method of the invention, in a patient that hasor is developing a biofilm, the particular microbial strain can beidentified prior to administration of the compound. As described in theExamples section, various strains of the same microbe may have avariation in the biofilm response to the presence of necrotic cells(e.g., neutrophils), as compared to other strains. The compound used toinhibit the accumulation of, necrosis of, or release of the cellularcontents of, cells that undergo necrosis can be selected or modifiedbased on this response, or the dosage or administration protocol may bedetermined or refined based on this response. For example, one may usethe patient isolate to identify compounds from a selection of compoundsthat are identified as being particularly useful to inhibit necroticcell-enhanced biofilm formation in the specific patient.

The methods of the present invention can be used in any animal (patient,subject, individual), and particularly, in any animal of the Vertebrateclass, Mammalia (i.e., mammals), including, without limitation,primates, rodents, livestock and domestic pets. Preferred mammals towhich the present method can be applied are humans.

Various aspects of the present invention are described in the followingexperiments. These experimental results are for illustrative purposesonly and are not intended to limit the scope of the present invention.

EXAMPLES

The following Materials and Methods were used in Examples 1-4 below.

P. aeruginosa and Neutrophil

P. aeruginosa used was strain PA01 (a motile piliated strain) or anisogenic strain of PA01 carrying the gene encoding GFP (31). Humanneutrophils were isolated from healthy volunteers, purified by theplasma Percoll method (21) and resuspended in RPMI 1640 (Bio-Whittaker,Walkersville, Md.) supplemented with 10 mM HEPES (pH 7.6) and 2%heat-inactivated platelet-poor plasma.

Biofilm Assays

A static biofilm assay was used with polypropylene tubes (9). PAO1 wasgrown overnight under constant rotation to late stationary phase at 37°C. in LB. All biofilm studies were initiated with neutrophils (1×10⁷cells/ml) and PA01 (1×10⁶ CFU/ml) in suspension, and cultures wereincubated under stationary conditions at 37° C. P. aeruginosa adherentto the tube was considered to be a biofilm, while bacteria not adherentto the surface of the tube was considered to be “planktonic”. Viable P.aeruginosa biofilm and planktonic cells were quantified by sonicatingthe adherent bacteria in LB, followed by serial dilution and plating todetermine CFUs on Pseudomonas Isolation Agar (Difco, Sparks, Md.). P.aeruginosa biofilm density was quantified by crystal violet (CV)staining (9). The contribution of background staining of neutrophilcomponents, tubes and reagents were subtracted from depicted values.Neutrophils were lysed with 0.1% Tween 20 (BioRad, Hercules, Calif.) for30 min. Neutrophil lysates relatively depleted of F-actin were preparedby precipitating the F-actin using the F-actin/G-actin In Vivo BiochemKit (Cytoskeleton, Denver, Colo.). The total protein present in thewhole cell lysate and the F-actin depleted lysate was determined by theBradford Protein Assay (BioRad, Hercules, Calif.). Biofilmexopolysaccharide was determined by total carbohydrate assays aspreviously described (15).

P. aeruginosa Actin-Binding Assay

Purified G-actin extracted from rabbit skeletal muscle was purchased(Sigma, St. Louis, Mo.) as a lyophilized powder containing 2 mM Tris, pH8.0, 0.5 mM beta mercaptoethanol, 0.2 mM CaCl₂, 0.2 mM ATP, and wasredissolved in deionized water at a concentration of 3-4 mg/ml, whichmaintains the G-form of actin. G-actin was polymerized to F-actin byincubating at room temperature for 1 h in the presence of 50 mM KCl and2 mM MgCl₂ in sterile PBS. G-actin was also incubated at roomtemperature for 1 hr in the absence of KCl and MgCl₂ to preventpolymerization. After 1 hour, F-actin and G-actin were plated on a96-well microtitre plate and incubated overnight at room temperature.All wells were blocked with 1% BSA for 1 hr before adding the bacteria.P. aeruginosa was labeled with the intracellular fluorescent conjugatecarboxy-fluorescein diacetate, succinimidyl ester (CFDA SE) for 45 min(Vybrant CFDA SE Cell Tracer Kit, Molecular Probes, Eugene, Oreg.) andthen washed with PBS. Labeled P. aeruginosa (1×10⁶) were added to wellscoated with either F- or G-actin or PBS (with or without KCl and MgCl₂)controls. The plates were incubated for 4 hr at 37° C. and then washedcarefully with PBS. The fluorescent intensity of bound P. aeruginosa wasmeasured at 492/517 nm with a plate reader (Bio-Tek Instruments) and thequantity of P. aeruginosa present was determined by a standard curve andexpressed as a percent of the total number of P. aeruginosa added to thewell. Enhanced F-actin formation on the plates was confirmed by stainingwith yellow-green-fluorescent NBD phallacidin (Molecular Probes) withrelative quantification read at 465/536 nm. Equivalent quantities oftotal F- and G-actin on the plates were confirmed by exposure of thewells with a mouse anti-pan actin IgG antibody (NeoMakers, Fremont,Calif.) followed by secondary binding with an anti-mouse IgG globulinconjugated with horseradish peroxidase which then binds to theantibody-antigen complex. The excess conjugate was removed by washing,followed by the addition of chromogen/substrate tetramethylbenzidine(TMB) with H₂O₂ and read at 450/570 nm. The total quantity of proteinwas confirmed to be equivalent for all conditions, as measured by theBradford Assay (BioRad, Hercules, Calif.).

Purification of Granular Proteins and DNA

Genomic DNA was isolated from neutrophils and P. aeruginosa using theDNeasy Tissue Kit (Qiagen, Valencia, Calif.) according to themanufacturer's protocol. Actin and DNA polymerization was performed asdescribed previously (38). Effects of filament cleavage was tested byinitially treating the samples with 90 Kunitz units/ml of DNase I (43)and/or 200 nM gelsolin (Sigma) (42). Granule proteins were isolated fromhomogenized human neutrophils by differential centrifugation in adiscontinuous Percoll/sucrose gradient (34). Heavy and light granuleswere immediately suspended in RPMI media and added to P. aeruginosa.

Microscopy

Samples of sputa from CF patients chronically infected with P.aeruginosa were frozen in liquid nitrogen and stored at −20° C. At thetime of analysis, sputa were thawed and resuspended carefully in PBS(1:4 vol/vol ratio) containing 10⁷ CFU/ml of P. aeruginosa labeled withthe intracellular fluorescent conjugate (CFDA SE) as described in theactin-binding protocol. Samples were incubated for 4 hours at 37° C.,then dried on Superfrost/Plus microscope slides (Fisher Scientific) andstained for 15 min with 10 μl of 0.6 μM Alexa Fluor 546 Phalloidin(Molecular Probes), 10 μl of 0.2 μM DAPI, dilactate (Molecular Probes),to visualize F-actin and DNA. Visualization of actin (546/576 nm), DNA(358/461 nm), and P. aeruginosa (492/517 nm) were performed sequentiallyon the same field, followed by an “overlay” view at all 3 wavelengths.Neutrophil lysates were also combined with P. aeruginosa labeled withCFDA SE for 4 hrs at 37° C., and staining of F-actin and DNA wasconducted as described for CF sputa.

Analysis of P. aeruginosa Biofilms by Confocal Microscopy (CM)

GFP-PAO1 was cultured in 8-chamber polystyrene tissue culture treatedglass slides (Falcon, Becton Dickinson Labware, Franklin Lakes, N.J.)alone or with neutrophils. At 48 hrs biofilms were evaluated using aZeiss Axiovert 200M confocal microscope equipped with Slidebook imagingsoftware (Intelligent Imaging, Denver, Colo.). GFP-PAO1 was excited inthe FITC channel at 488/500-550 nm. 3-D reconstruction of biofilms wasformed from images captured at 1 μm intervals, with segmentation andreconstruction using version 3.5 SURFdriver software (Kailua, Hi.).

Statistical Analysis

Data were analyzed using JMP software (SAS Institute, Cary, N.C.).Student's unpaired t test (two-tailed) was use to determine significanceof neutrophil and neutrophil lysate enhancement of P. aeruginosasurvival and biofilm development (FIGS. 1A-C, 3A) and binding of P.aeruginosa to F-actin (FIG. 4) at individual time points. One-way ANOVAusing Dunnett's Method was used to analyze variance of multiple groupmeans to the control group (P. aeruginosa alone) for biofilm development(FIGS. 1D-E, 2A-D, 3B-C). For all tests, p<0.05 was consideredsignificant.

Example 1

The following example demonstrates the effect of human neutrophils oninitial P. aeruginosa biofilm development.

Referring to FIG. 1A, biofilm development of P. aeruginosa (□) wascompared with P. aeruginosa in the presence of neutrophils (●).Neutrophil cytotoxicity (▴) equaled 92% after 24 hrs of exposure to P.aeruginosa (FIG. 1A; lot (right axis) depicts mean percent of viableneutrophils±SD (n=4)). In the presence of neutrophils, a reduction inthe number of surviving P. aeruginosa was detected at 4 hours, while atlater timepoints, the bactericidal effects of the neutrophil no longerreached significance (FIG. 1A; plot depicts mean±SD of CFU (n=4).*p<0.05 by Student's t-test).

When measured simultaneously, the number of viable P. aeruginosa in theplanktonic state was significantly decreased by the presence ofneutrophils, while the number of viable P. aeruginosa in the biofilmstate was significantly increased (FIGS. 1B-C). Referring to FIG. 1B,the presence of neutrophils (●) resulted in fewer viable P. aeruginosain the planktonic state compared to P. aeruginosa in the absence ofneutrophils (□) (plot depicts mean±SD of CFU (n=4). *p<0.05 by Student'st-test). Referring to FIG. 1C, neutrophils (●) increased the number ofviable P. aeruginosa in the biofilm state compared to P. aeruginosaalone (□) when measured simultaneously with P. aeruginosa in theplanktonic state shown in FIG. 1B (plot depicts mean±SD of CFU (n=4).*p<0.05 by Student's t-test).

CV staining of P. aeruginosa biofilms formed in the presence ofneutrophils demonstrated an increase in biofilm density (FIG. 1D; plotdepicts mean±SD of O.D. measurements (n=21). *p<0.05 by Dunnett'st-test). Specifically, neutrophils (●) increased biofilm density(assayed by CV staining) compared to P. aeruginosa alone (□) by 4 hours.

Enhanced biofilm formation in the presence of neutrophils was alsodemonstrated by quantifying bacterial exopolysaccharide production (FIG.1E; plot depicts mean±SD of O.D. measurements (n=21). *p<0.05 byDunnett's t-test). Specifically, exopolysaccharide staining of biofilmdensity demonstrated that the presence of neutrophils (●) resulted in agreater quantity of biofilm compared to P. aeruginosa in the absence ofneutrophils (□) by 4 hrs.

As neutrophils are recruited continuously to the CF airway, the effectof adding additional viable neutrophils 24 and 48 hrs after initiationof biofilm formation was tested. Supplementing neutrophils to P.aeruginosa in the early stages of biofilm development resulted inadditional biofilm enhancement (FIGS. 1D-E). When additional neutrophilswere added 24 and 48 hrs (arrows) after the initiation of the biofilm,further enhancement of the biofilm density (♦) was observed at 48 and 72hrs.

For each assay described above, significant enhancement of biofilmdevelopment was observed by 4 hours, and by 72 hours the extent ofneutrophil-induced biofilm enhancement exceeded 3.5-fold as assessed byviable bacterial colony counts, 2.5-fold as assessed by biofilm density,and 2-fold as assessed by exopolysaccharide content.

Confocal microscopy and 3-D reconstruction of GFP-labeled P. aeruginosabiofilms in the presence of neutrophils depicted a thicker and moredeveloped biofilm, when compared to the absence of neutrophils (data notshown). Together, data presented in FIGS. 1 and 2 demonstrate thepotential of the neutrophil to enhance the earliest stages of P.aeruginosa biofilm formation.

Example 2

The following example shows the enhancement of P. aeruginosa biofilmformation by lysed neutrophils.

Since neutrophil necrosis is rapid in the presence of P. aeruginosa, thecapacity of the cellular content of lysed neutrophils to evoke enhancedP. aeruginosa biofilm development was tested. Parameters of biofilmdevelopment of P. aeruginosa (□) were compared with P. aeruginosa in thepresence of lysed neutrophils (●) at time intervals from 0 to 72 hrs.Combining P. aeruginosa with neutrophil lysates significantly enhancedbiofilm formation as measured by CV staining and exopolysaccharidesynthesis, and supplementing the early biofilm with additionalquantities of lysed neutrophils at 24 and 48 hrs further enhancedbiofilm production (FIGS. 2A,C). Referring to FIG. 2A, crystal violetstaining of biofilm density demonstrated that the presence of lysedneutrophils resulted in a greater quantity of biofilm compared to P.aeruginosa in the absence of neutrophils by 4 hrs. When additionalneutrophil lysates (♦) were added 24 and 48 hrs (arrows) after theinitiation of the biofilm, further enhancement of the biofilm densitywas observed at 48 and 72 hrs. Referring to FIG. 2C, exopolysaccharidestaining of biofilm density demonstrated that the presence of lysedneutrophils resulted in a greater quantity of biofilm compared to P.aeruginosa in the absence of neutrophils by 4 hrs. When additionalneutrophil lysates (♦) were added 24 and 48 hrs (arrows) after theinitiation of the biofilm, further enhancement of the biofilm densitywas observed at 48 and 72 hrs.

Biofilms formed in the presence of lysed neutrophils achieved 92% of thebiofilm enhancement of an equivalent number of viable neutrophils whenassayed by CV staining, and 94% percent when assayed byexopolysaccharide synthesis (FIGS. 2B,D). Furthermore, the number ofviable surface attached biofilm cells increased when lysed neutrophilswere added, while the number of viable planktonic cells decreased (datanot shown). Referring to FIG. 2B, crystal violet staining of biofilmdensity demonstrated that by 72 hrs, lysed neutrophils added at 0, 24,and 48 hrs (hatched bar) achieved 92% of the biofilm development seenwith viable neutrophils added at 0, 24, and 48 hrs (solid bar). Bothconditions resulted in significantly greater biofilm development whencompared to P. aeruginosa in the absence of neutrophils (open bar).Referring to FIG. 2D, exopolysaccharide staining of biofilm densitydemonstrated that by 72 hrs, lysed neutrophils added at 0, 24, and 48hrs (hatched bar) achieved 94% of the biofilm development seen withviable neutrophils added at 0, 24, and 48 hrs (solid bar). Bothconditions resulted in significantly greater biofilm development whencompared to P. aeruginosa in the absence of neutrophils (open bar).Plots A-D depict mean±SD of O.D. measurements (n=21). *p<0.05 byDunnett's t-test.

Example 3

The following example demonstrates the enhancement of P. aeruginosabiofilm formation by isolated neutrophil components.

The data described in the Examples above indicates that neutrophilcellular contents are largely responsible for enhanced P. aeruginosabiofilm formation. Analysis of CF sputum demonstrates highconcentrations of granule proteins, actin and DNA released from necroticneutrophils (18, 28, 38). The capacity of each of these compounds tomediate enhanced early P. aeruginosa biofilm formation was tested.Referring to FIG. 3A, P. aeruginosa (▾) was combined with granuleproteins (quantity equivalent to 5×10⁶ neutrophils) compared to P.aeruginosa (□) in the absence of granule proteins and P. aeruginosacombined with live neutrophils (●). Supplementing planktonic P.aeruginosa with purified granule proteins failed to enhance biofilmproduction over a range of concentrations (FIG. 3A; Plot depicts mean±SDof O.D. crystal violet measurements (n=4). *p<0.05 by Student's t-test).

Neutrophil actin and DNA are also abundant in CF sputum and have beenobserved to bind together, forming polymers that increase the viscosityof CF sputum (38). Supplementing purified globular monomeric actin(G-actin (▴); 0.4 mg/ml) to P. aeruginosa under conditions known toresult in formation of actin filaments (F-actin) significantly enhancedbiofilm formation by 72 hrs (FIG. 3B; plot depicts mean±SEM of O.D.measurements of CV staining (n=4)).

Purified neutrophil DNA alone (X; 4 ug/ml) did not enhance P. aeruginosabiofilm production. However, supplementing planktonic P. aeruginosa withboth actin and neutrophil DNA (♦) achieved an enhancement of biofilmformation, equaling 88% of the biofilm developed in the presence of liveneutrophils (●) by 72 hrs. An equivalent effect on biofilm developmentwas observed using DNA isolated from P. aeruginosa instead ofneutrophils (data not shown); however, DNA found within CF sputa isalmost entirely of human origin (28). Neutrophil lysates relativelydepleted of F-actin and adjusted to an equal protein concentration asthe whole cell lysates were also found to result in a significantdecreased in biofilm enhancement when compared to the untreated wholecell lysates (data not shown). Addition of actin or actin with DNA to P.aeruginosa was significantly greater then P. aeruginosa alone at times4-72 hrs (*p<0.05 by Dunnett's t-test).

A recent study reported that extracellular DNA (originating from P.aeruginosa) is required for the initial establishment of P. aeruginosabiofilms, and addition of DNase strongly inhibited biofilm formation(43). The addition of DNase abolished much of the neutrophil-inducedenhancement of biofilm formation (FIG. 3C; plot depicts mean±S.D. ofO.D. CV measurements (n=4). *p<0.05 by Dunnett's t-test) withoutsignificantly inhibiting bacterial growth or neutrophil survival (datanot shown). The addition of gelsolin, a protein that severs noncovalentbonds between monomers of actin filaments also significantly reduced theneutrophil-induced enhancement of the biofilm, but to a much lesserextent than DNase (FIG. 3C). Although as a single component, theaddition of purified actin evoked the greatest biofilm enhancement (FIG.3C), it must be noted that detectable amounts of DNA (originating fromP. aeruginosa) were present in the absence of added DNA or neutrophils(data not shown). Likewise, while DNase evoked the greatest reduction inbiofilm enhancement (FIG. 3C), the enzyme can bind to monomeric actinand slowly depolymerize actin filaments (22).

Purified neutrophil DNA (in the absence of actin) forms small fragmentswith no apparent association with planktonic P. aeruginosa (data notshown). In the absence of exogenous DNA, actin formed filaments ofvarying size, and P. aeruginosa appeared associated with these filaments(data not shown). The combination of purified actin and DNA resulted inrobust filament formation, with virtually all visible P. aeruginosaattached to the polymer (data not shown). The inventors devised an assayto test for binding of P. aeruginosa to actin.

Referring to FIG. 4, planktonic P. aeruginosa was allowed to settle inwells coated with actin (solid bar), or BSA-blocked plastic (open bar).After 4 hours of incubation, 44% of P. aeruginosa was bound to F-actin,in comparison to 11% of P. aeruginosa which bound to G-actin, which wasnot different then albumin-coated plastic (FIG. 4; Plot depicts means±SD(n=3); P<0.05 by Student's t test).

Example 4

The following example shows P. aeruginosa association with actin/DNApolymers from neutrophils and in CF sputa.

Immunofluorescence of necrotic neutrophils stained for actin and DNA,revealed co-localization of both components (data not shown), andconfirmed the formation of actin-DNA filaments in CF sputa, aspreviously reported (38). In both neutrophil lysates and CF sputa, P.aeruginosa localized primarily to actin-DNA filaments after 4 hours ofincubation (data not shown), supporting the concept that these fibersprovide a matrix for initial P. aeruginosa attachment and biofilmestablishment. Treatment of both the neutrophil lysates and CF sputumwith DNase I resulted in near complete disruption of the actin-DNAfilaments and dispersion of the P. aeruginosa, with a greater number ofvisually observable planktonic bacterial cells (data not shown).

Example 5

The following example shows that, in the presence of neutrophils, P.aeruginosa forms multicellular aggregates.

While the present inventors have observed environmental strains of P.aeruginosa and PA01 do not commonly undergo autoaggregation, in CFsputum, the bacteria was found to be present only in the form ofmulticellular clusters. Over time, a small colony variant (SCV)phenotype can evolve in the CF airway that is associated with a highdegree of autoaggregation, as well as increased virulence. When combinedwith neutrophils, PA01 induces rapid necrosis of the neutrophils (Walkeret al., 2005, Infect Immun 73(6):3693-701), and concurrently, thebacteria is seen to form loose clusters of cells (data not shown).Although the inventors have shown that P. aeruginosa binds toneutrophil-derived actin and DNA to enhance biofilm growth, it ispossible that this mechanism also serves to allow environmental or earlyCF strains to aggregate in the CF airway.

Example 6

The following example demonstrates P. aeruginosa response to humanneutrophils is determined by P. aeruginosa gene expression.

Although one mechanism by which neutrophils enhance P. aeruginosabiofilm development is by providing a scaffolding of actin and DNApolymers, it is probable that other ligands are involved, and thereceptors used by the bacteria are unknown. The inventors testedneutrophil-induced biofilm enhancement of PA01 to a number of isogenicmutant strains lacking expression of various gene products implicated inP. aeruginosa mediated lung disease. Significant differences in theextent of response to neutrophils were observed in the mutant strains.Deletion of the genes encoding the quorum-sensing signals rhl (ΔrhlR),las (ΔlasR) or both (ΔrhlR/lasR) resulted in little change in biofilmdevelopment in the absence of neutrophils and a significant reduction inthe neutrophil-induced biofilm enhancement compared to unmodified PA01(ie., these mutant strains did not respond to neutrophils by developinga thicker biofilm) (FIG. 5).

PvdS is an extracytoplasmic function signal factor which is required forthe production of pyoverdine, exotoxin A and PrpL protease, andfunctions as well to coordinate the response of the bacteria to ironstarvation. Deletion of the gene pvdS has been associated with decreasedbiofilm development. However, deletion of the gene encoding PvdS (ApvdS)resulted in a robust neutrophil-induced biofilm enhancement within 4hours, far exceeding the response of unmodified PA01 (not shown). At 72hrs, the response of ΔpvdS equaled that of PA01 (FIG. 6). TatC is a genethat encodes for the TAT pore apparatus the regulates secretion of manyproteins which determine P. aeruginosa virulence, as well as motilityand biofilm formation (Ochsner et al., 2002, Proc Natl Acad Sci USA99:8312). Deletion of tatC (ΔtatC) resulted in slightly reduced biofilmformation which was not effected by the presence of neutrophils (i.e.,showed an absence of neutrophil-induced enhancement) (FIG. 6).

As previously observed, mucoid strains of P. aeruginosa demonstraterelatively decreased biofilm formation on abiotic surfaces. Two mucoidstrains that are alginate overproducers, an isogenic mutant of PA01 withdeletion of mucA (ΔmucA) and a well-characterized CF strain (FRD1) alsoresulted in decreased biofilms compared to unmodified PA01. However, inthe presence of human neutrophils, the ability of these mutants toproduce biofilms was restored, essentially nullifying the phenotypicchanges induced by the genetic modification (FIG. 7). As the ability ofthe mutant strains to form biofilms differs, the effect of neutrophil onmodifying biofilm enhancement is best appreciated when calculated as aratio compared to bacteria alone. FIG. 8 provides an index ofneutrophils-enhancement of mutant strains, where the density of thebiofilm in the presence of neutrophils is plotted as a fold-increase ofthe biofilm density relative to the strain in the absence ofneutrophils. FIG. 8 shows the fold-change evoked by neutrophils, whichranges from 1.1 for ΔrhlR/lasR to 5.1 for FRD1. Together, FIGS. 5-8support the conclusion that the extent of P. aeruginosa response tohuman neutrophils is determined, in part, by bacterial gene expression.

Example 7

The following example demonstrates that P. aeruginosa binding to F-actinis determined by P. aeruginosa gene expression.

The inventors have devised an assay to test the ability of P. aeruginosato bind to actin (see Example 3), and found significant binding of thebacteria to actin filaments (F-actin), but not to an equal quantity ofpurified globular monomeric actin (G-actin) following 4 hours ofincubation. The inventors tested selected isogenic mutants of PA01 fordifferences incapacity to bind to F-actin. Considerable heterogeneitywas observed in the ability of these mutants to bind F-actin (FIG. 9A;*p<0.05 by Dunnetts test of multiple comparisons). Of particularimportance, binding of PA01 and isogenic mutants to F-actin was found tosignificantly correlate to the fold-change of neutrophil-induced biofilmenhancement (FIG. 9B; R2=0.85, p=0.0025). Thus, the mutant which boundF-actin to the greatest extent also had the greatest relativeenhancement in biofilm development in the presence of neutrophils.

Example 8

The following example demonstrates that human neutrophils selectivelymodify antibiotic resistance of P. aeruginosa biofilms.

As human neutrophils enhance biofilm development, the inventorsquestioned if biofilms formed in the presence of neutrophils had alteredpatterns of antibiotic resistance. Biofilms and planktonic P. aeruginosagrown in the presence or absence of neutrophils for 24 hours were testedfor susceptibility to four clinically relevant classes of antibiotics.Despite greater thickness, biofilms grown in the presence of neutrophilsdemonstrated significantly greater susceptibility to both tobramycin andciprofloxacin, while susceptibility to azithromycin and ceftazidime wasunchanged (Table 1). No significant changes in antibioticsusceptibilities was detected in planktonic P. aeruginosa in thepresence of neutrophils. This selective modification of antibioticresistance supports the conclusion that neutrophils mediate phenotypicchanges in 5 addition to greater biofilm thickness. TABLE 1 Antibioticsusceptibility of P. aeruginosa strain PA01 as a planktonic population(MIC) and as a biofilm population (MBEC) as derived by the NCCLS assayMIC (μg/ml) MBEC (μg/ml) Antibiotic PA01 PA01 + PMN PA01 PA01 + PMNAzithromycin 64 32 >1024 1024 Ceftazidime 2 2 >1024 >1024 Ciprofloxacin0.5 2 512 128 Tobramycin 1 0.5 >1024 256

Example 9

The following examples shows the effects of mucin on neutrophilmodification of biofilm growth.

When P. aeruginosa was suspended in mucin rather then RPMI media, asimilar effect was observed on NUNC-TSP biofilm growth. In the absenceof neutrophils, mucin increased biofilm growth, while in the presence ofneutrophils no change was detected (FIG. 10; *p<0.05 by Student'st-test). Of even greater interest, microscopic examination of P.aeruginosa suspended within the mucin demonstrated the presence of smallbacterial aggregates, that were markedly increased in size by thepresence of neutrophils (data not shown). These aggregates of P.aeruginosa may represent the predominant structure of biofilms that arepresent in the CF airway, and the combination of neutrophils with mucinappears to enhance their formation.

Example 10

The following examples shows neutrophil-induced enhancement of Bccbiofilm growth.

Bcc represents a clinically significant infection in a subset of CFpatients. The inventors questioned if neutrophils could also enhance thebiofilm development of Bcc, which forms biofilms poorly in the NUNC-TSPsystem. In the presence of neutrophils, a selective enhancement ofspecific strains was observed by 24 hours (FIG. 11). Of considerableinterest, some Bcc strains exhibited relatively small neutrophilenhancement, and the biofilm development of one strain (BD AU0645strain) was inhibited in the presence of neutrophils. The tremendousheterogeneity in response to neutrophils by clinical strains of Bccsupports the conclusion that specific mechanisms regulateneutrophil-induced biofilm enhancement, and that this variability mayalso be present between various CF strains of P. aeruginosa. Althoughnot tested in this experiment, the heterogeneity in neutrophil-inducedbiofilm development of Bcc could represent a portion of the variabilityin virulence of this organism in CF lung disease.

Example 11

The following example describes a novel model of chronic P. aeruginosainfection in the murine airway.

Although the agar-bead model reproduces many important features ofinfection in the CF airway, it is designed primarily to study theresponse of the lung to P. aeruginosa, and not the response of P.aeruginosa to the innate immune system. Specifically, immobilizing P.aeruginosa on beads or catheters bypasses mechanisms required for earlybiofilm formation, including interactions between bacterial adhesionsand polymers originating from the host. Based on current theories of P.aeruginosa biofilm formation in the CF airways, and the inventors' ownfindings in vitro, it was believed that a number of components arerequired to better replicate P. aeruginosa biofilm formation in vivo.

1) Infection with low numbers of wild-type P. aeruginosa, that has notacquired virulence factors such as mucoidy. Although high concentrationsof mucoid P. aeruginosa are eventually present in CF lung disease, theinitial infection occurs with low numbers of relatively avirulentenvironmental strains of P. aeruginosa (Burns et al., 2001, J Infect Dis183:444).

2) Trapping of bacteria in abnormal airway surface fluid and secretions(Boucher et al., 2002, Adv Drug Delivery Res 54:1359).

3) Reduced ability to clear the trapped bacteria.

4) Excessive and persistent neutrophil recruitment to the airways. Thevast array of cytotoxic compounds released by activated neutrophils isclearly implicated in airway damage in CF.

5) The presence of cellular debris from necrotic neutrophils. Sputumplugs, largely comprised of necrotic neutrophils, have been identifiedas the location of biofilm existence in the airway, and elements ofblood including RBCs, serum proteins, clotting factors, complement,platelets, are all commonly present in CF sputum.

6) Obstruction of the airway is an early and central feature in CF andis likely essential in the pathogenesis of bronchiectasis.

To achieve these features, the inventors transtracheally instilled P.aeruginosa (5×10³ of PA01) suspended in purified thrombin into a distalmouse bronchus, followed by human plasma (also containing RBCs andplatelets). In the presence of plasma, thrombin induces rapid clottingof fibrinogen in situ, resulting in a focal airway plug comprised ofelements present in the CF airway, and infected with low numbers of P.aeruginosa. Although mice subjected to this procedure demonstratedbehavior consistent with an acute inflammatory insult for the first24-48 hours, their appearance subsequently returned to baseline.Following 7 and 14 days post-infection, mice were sacrificed foranalysis of infection, inflammation, and histologic changes of the lung.At day 7, P. aeruginosa infection in the lung (n) was below the initialinoculum, with evidence of dissemination to the spleen (c). However, byday 14, the burden of P. aeruginosa had increased by almost 2 logs(compared to the quantity installed at day 0), and the infection wascleared from the spleen (data not shown). Leukocyte accumulation to theairway (assessed by BAL), was robust at day 7 and persistent at day 14,while animals receiving plasma and thrombin in the absence of bacteriareturned to baseline levels (data not shown). Lung histology (by H&Estaining) demonstrated intense areas of focal lung inflammation (datanot shown), with scattered clusters of “WBC clots” within the airways(data not shown). These airway plugs may represent a site of persistentinfection, based on findings from the CF lung. However, microcolonies ofP. aeruginosa, or classic features of bronchiectasis, were not apparentat 14 days.

Each publication cited herein is incorporated herein by reference in itsentirety.

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While various embodiments of the present invention have been describedin detail herein, it is apparent that modifications and adaptations ofthose embodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims:

1. A method to inhibit biofilm formation or reduce biofilms in asubject, comprising administering to a subject that has or is at risk ofdeveloping biofilms, a compound that inhibits the formation orpolymerization of actin microfilaments or depolymerizes actinmicrofilaments at or proximal to the site of biofilm formation or thesite of infection by a microorganism that forms biofilms.
 2. The methodof claim 1, wherein the actin microfilaments are formed from the contentof a cell that undergoes necrosis at or proximal to the site of biofilmformation or the site of infection by a microorganism that formsbiofilms.
 3. The method of claim 2, wherein the cell is a neutrophil. 4.The method of Clam 1, further comprising administering to the subject ananti-DNA compound.
 5. The method of claim 1, further comprisingadministering to a subject a compound that inhibits accumulation of,inhibits necrosis of, or inhibits release of the cellular contents of,cells that undergo necrosis, at or proximal to the site of biofilmformation or the site of infection by a microorganism that formsbiofilms.
 6. The method of claim 5, wherein the cells that undergonecrosis are neutrophils.
 7. The method of claim 6 wherein the compoundinhibits the adherence of, migration to, or the sensing or response tochemoattractants by neutrophils, or inhibits the activity or release ofa cytokine, chemokine or chemoattractant that attracts or enhancesneutrophil activity.
 8. The method of claim 5, wherein the compound isan anti-inflammatory compound.
 9. The method of claim 5, furthercomprising administering to the subject an anti-DNA compound.
 10. Themethod of claim 1, further comprising administering to the subject ananti-mucin compound.
 11. The method of claim 1, further comprisingadministering to the subject a compound for treatment of a disease orcondition associated with biofilm formation.
 12. The method of claim 1,wherein the compound is administered when a disease or conditionassociated with biofilm formation is diagnosed or suspected.
 13. Themethod of claim 1, wherein the compound is administered prior to thetreatment of the subject with a process that may cause a biofilm to formin the patient.
 14. The method of claim 1, wherein the compound isadministered with a pharmaceutically acceptable carrier.
 15. The methodof claim 1, wherein the compound is administered directly to or proximalto the site of biofilm formation or potential therefore.
 16. The methodof claim 1, wherein the compound is administered to the lung or airwaysof the subject.
 17. The method of claim 1, wherein the compound isapplied to a prosthetic graft or administered to the subject receivingthe graft prior to or during the implantation or utilization of thegraft.
 18. The method of claim 1, wherein the compound is applied to acatheter prior to or during use of the catheter by a subject.
 19. Themethod of claim 1, wherein the compound is applied to the site of awound or to the wound dressing when the wound is treated.
 20. The methodof claim 1, wherein the compound is applied to a medical device thatcontacts a subject tissue surface prior to or during use of the medicaldevice by a subject.
 21. The method of claim 1, wherein the biofilmforms in connection with a disease or condition in an organ, tissue orbody system.
 22. The method of claim 1, wherein the biofilm forms on asurface of a tissue, organ or bodily part.
 23. The method of claim 1,wherein the biofilm forms in connection with a disease or conditionselected from the group consisting of: infectious kidney stones,cystitis, catheter-related infection (kidney, vascular, peritoneal),medical device-related infections, prostatitis, dental caries, chronicotitis media, cystic fibrosis, bronchiectasis, bacterial endocarditis,Legionnaire's disease, orthopedic implant infection, osteomyelitis,wounds, acne, and biliary stents.
 24. The method of claim 1, wherein thesubject has or is suspected of having cystic fibrosis.
 25. The method ofclaim 1, wherein the microorganism is Pseudomonas aeruginosa.
 26. Amethod to inhibit biofilm formation or reduce biofilms in a subject,comprising administering to a subject that has or is at risk ofdeveloping biofilms: (1) a first compound that inhibits the formation orpolymerization of actin microfilaments or depolymerizes actinmicrofilaments; and (2) an anti-DNA compound, wherein the first compoundand the anti-DNA compound are administered at or proximal to the site ofbiofilm formation or the site of infection by a microorganism that formsbiofilms.
 27. A composition for inhibiting biofilm formation or reducingbiofilms in a subject, comprising: (1) a first compound that inhibitsthe formation or polymerization of actin microfilaments or depolymerizesactin microfilaments at or proximal to the site of biofilm formation;and (2) a second compound that is an anti-DNA compound.
 28. Thecomposition of claim 27, further comprising a compound that inhibits theaccumulation of, necrosis of, or release of the cellular contents of,neutrophils at or proximal to the site of biofilm formation, and acarrier suitable for application to the site of biofilm formation.
 29. Acomposition for inhibiting biofilm formation or reducing biofilms in asubject, comprising: (1) a first compound that inhibits the formation orpolymerization of actin microfilaments or depolymerizes actinmicrofilaments at or proximal to the site of biofilm formation; and (2)a second compound that inhibits the accumulation of, necrosis of, orrelease of the cellular contents of, neutrophils at or proximal to thesite of biofilm formation, and a carrier suitable for application to thesite of biofilm formation.
 30. A method to identify a compound thatinhibits necrotic cell-enhanced biofilm formation or reduces necroticcell-enhanced biofilms in a subject, comprising: a) contacting aputative inhibitory compound with a microbial culture in the presenceand absence of a population of cells or a lysate thereof, wherein themicrobial culture forms biofilms and is in a planktonic state prior tocontact with the putative inhibitory compound, and wherein thepopulation of cells undergoes necrosis in the presence of the microbialcells; and b) measuring biofilm formation after contact with theputative regulatory compound as compared to in the absence of thecompound and as compared to in the presence and absence of thepopulation of cells or lysate thereof; wherein a decrease in biofilmformation in the presence of the putative regulatory compound and thepresence of the population of cells or lysate thereof, as compared to inthe absence of the population of cells or lysate thereof and as comparedto in the absence of the putative regulatory compound, indicates thatthe putative regulatory compound inhibits necrotic cell-enhanced biofilmformation or reduces necrotic cell-enhanced biofilms.
 31. The method ofclaim 30, wherein the population of cells is a population ofneutrophils.